Pentacyclic triterpene compounds and uses thereof

ABSTRACT

Disclosed herein is a PEGylated bis pentacyclic triterpene, compositions comprising the PEGylated bis pentacyclic triterpene, methods of preparing the PEGylated bis pentacyclic triterpene, and a method of treating or preventing a fungal disease in a plant using the compounds and compositions disclosed herein. The PEGylated bis pentacyclic triterpene has the formula A-P-B, wherein A is a first pentacyclic triterpene; B is a second pentacyclic triterpene; and P is a polyethylene glycol (PEG) molecule.

FIELD

The present disclosure relates generally to compounds comprising apentacyclic triterpene, for use in preventing or treating fungaldiseases in plants.

BACKGROUND

Fungal and fungal-like pathogens are the cause of many common plantdiseases and result in losses in agricultural yield, quality and profit.Blight is an example of a plant disease that is caused by fungal orfungal-like organisms and is one of the most destructive disease ofpotato in Canada and worldwide.

Current agents or fungicides used to treat fungal infections includepolyene antibiotics, synthetic azoles and griseofulvin. Repeated use ofsuch fungicides results in the development of resistance by thepathogen. When fungicide resistance develops, the product or otherchemically similar products no longer controls the disease effectively.Thus, new, low-toxic fungicides belonging to different chemical groupsare needed to maintain control of damaging diseases caused by fungi andfungal-like organisms.

Pentacyclic triterpences such as Betulin (lup-20(29)-ene-3β,28-diol),Betulinic acid and lupeol (3β, 13ξ)-Lup-20(29)-en-3-ol) are abundant,naturally occurring triterpenes that can be isolated from plants such aswhite birch bark. Extracts from white birch bark have been used to treatinflammations, hepatitis, lymphatic disorders, tuberculosis cancers, andskin irritations. Moreover, pentacyclic triterpenes have been shown tohave anti-viral activity against herpes simplex virus (U.S. Pat. No.5,750,578) and to have anti-bacterial and anti-fungal activity. Forexample, WO 02/26761 A1 discloses the use of betulin and its derivativesas an anti-fungal agent against human pathogenic fungi Microsporumcanis, Microsporum audoinii, Trichophyton mentagrophytes, Epidermophytonfloccosum in in vitro growth inhibition assays performed on agar slantsand in liquid cultures.

U.S. Pat. No. 6,458,834 discloses compositions comprising pentacyclictriterpenes including ursolic acid, betulin and betulinic acid for useagainst the plant pathogenic fungi Phytopthora infestans, Alternariasolani, Botrytis cinerea and Cersospora arachidola.

Currently, there is a need for new anti-fungal compositions that includepentacyclic triterpenes. A need particularly exists for compositionsthat are effective against blight.

SUMMARY

It is an object of the present disclosure to obviate or mitigate atleast one disadvantage of previous antifungal compositions comprisingpentacyclic triterpenes.

Disclosed herein is a PEGylated bis pentacyclic triterpene having theformula: A-P-B, wherein A is a first pentacyclic triterpene; B is asecond pentacyclic triterpene; and P is a polyethylene glycol (PEG)molecule, for use in preventing or treating a fungal disease in a plant.

In an embodiment, the first and second pentacyclic triterpenes are eachindependently betulin, betulinic acid, lupeol, or an analogue orderivative thereof. For example, the first and second pentacyclictriterpene may each be betulin.

Also disclosed herein is a fungicidal composition comprising thePEGylated bis pentacyclic triterpene described herein, and anagriculturally acceptable diluent.

Also described herein is a method of treating or preventing a fungaldisease in a plant comprising: applying to the plant, to a soilsupporting the plant, or to both the plant and the soil supporting theplant, a composition comprising a PEGylated bis pentacyclic triterpenehaving the formula: A-P-B, wherein A is a first pentacyclic triterpene;B is a second pentacyclic triterpene; and P is a polyethylene glycol(PEG) molecule; and an agriculturally acceptable diluent.

Also described herein is a method of preparing a PEGylated bispentacyclic triterpene, the method comprising: combining a polyethyleneglycol (PEG) molecule with an organic solvent to form a mixture; heatingthe mixture to reflux; removing the distillate formed in the heatingstep from the mixture; adding two stoichiometric equivalents of apentacyclic triterpene with respect to the PEG molecule, a condensingagent, and, optionally, an acyl transfer catalyst to the mixture;stirring the mixture; cooling the mixture; and drying the mixture. Thepentacyclic triterpene may be betulin, betulinic acid, lupeol, or ananalogue or derivative thereof. The organic solvent may be toluene. Thecondensing agent may be N,N′-diisopropylcarbodiimide (DIC), and the acyltransfer catalyst, if required, may be dimethylaminopyridine (DMAP).

Also disclosed is a PEGylated bis pentacyclic triterpene prepared bythis method, and the use of the compound so prepared for preventing ortreating a fungal disease in a plant by applying the compound to theplant, to a soil supporting the plant, or to both the plant and the soilsupporting the plant.

Other aspects and features will become apparent to those ordinarilyskilled in the art upon review of the following description of specificembodiments in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will now be described, by way ofexample only, with reference to the attached Figures.

FIG. 1 is a graph showing the mean number of late blight lesions perplant (AUDPC).

FIG. 2 is a graph showing the mean percentage of diseased leaves perplant (AUPDC).

FIG. 3 is a graph showing the mean percentage of leaf area diseased perplant (AUDPC).

FIG. 4 is a graph showing the mean percentage of leaf area diseased perplant per week.

DETAILED DESCRIPTION

Pentacyclic triterpenes such as betulin and its analogues andderivatives are hydrophobic compounds and are poorly soluble in water.Because of their low water solubility, the handling and administrationof pentacyclic triterpenes is difficult as they are difficult to applyto crops in non-emulsion formulations. As a result, the use of betulinand its analogues and derivatives as antifungal agents has been limited.

One strategy for increasing the solubility of water insoluble compoundsis to make conjugates of a pro-drug with a high molecular weightpolyethylene glycol (PEG) molecule. Various in vivo experiments haveestablished that the molecular weight of PEG for these types ofpro-drugs must be greater than 40 kDa in order to maintain an adequateresident circulation time and allow for an efficacious therapeuticeffect.

The inventors have identified that the use of high molecular weight PEGs(>40 KDa) to produce water soluble pro-drugs is not necessary foragricultural efficacy and that pentacyclic triterpenes that are slightlysoluble are more effective at treating or preventing fungal infections.The inventors have surprisingly found that PEGylated bis pentacyclictriterpenes are more effective as anti-fungal agents than PEGylated monobis pentacyclic triterpenes. Without being bound by theory, it isbelieved that high solubility does not necessarily correlate with highactivity with respect to PEGylated bis pentacyclic triterpenes, such as,for example, PEGylated bis betulin.

Generally, the present disclosure provides compounds and compositionsthat are useful in preventing or treating fungal diseases in plants.Specifically, the disclosed compounds are PEGylated bis pentacyclictriterpenes having the formula:

A-P-B

wherein, A and B are each independently pentacyclic triterpenes and P isa polyethylene glycol (PEG) molecule that forms a covalent bond with Aand B.

The compositions disclosed herein comprise compounds of the formulaA-P-B and an agriculturally acceptable diluent.

The term “pentacyclic triterpenes” is meant to describe members of theterpene family that consist of six isoprene units (arranged in 5 rings)and have the molecular formula C₃₀H₄₈. Pentacyclic triterpenes areeasily isolable compounds and are usually concentrated in the outermostlayers of a plant such as the plant cuticle, fruit peel or bark.Terpenes are a large and diverse class of organic compounds, produced bya variety of plants, particularly conifers and are derivedbiosynthetically from units of isoprene, which have the molecularformula C₅H₈ and are classified by the number of isoprene units in themolecule. Advantageously, the anti-fungal compounds and compositionsdisclosed herein are not very expensive to manufacture as they areeasily extracted from abundant natural products or are easilysynthesized.

Pentacyclic triterpenes can be classified into many subgroups includinglupane, oleanane, gammacerane, hopane or ursane groups based on theircarbon skeleton. Lupanes have the following general structure:

In some embodiments, the pentacyclic triterpenes are members of thelupane group. Members of the lupane group include, but are not limitedto, lupenone, lupeol, betulin, betulinic acid, and tiarellic acid.Lupanes have been extensively studied as a natural source oftherapeutics. Most lupane-type terpenoids cholesterol-like triterpenoidsare virtually insoluble in water and exhibit poor phamacogenicproperties.

Betulin

Betulin, which is also known as lup-20(29)-ene-3β,diol, has themolecular formula C₃₀H₅₀O₂ and is an abundant, naturally occurringtriterpene of lupane structure. It has a pentacyclic ring structure, andhydroxyl groups in positions C3 and C28. Betulin is commonly isolatedfrom the bark of birch trees and forms up to 30% of the dry weight ofthe extractive.

Betulinic Acid (3β-hydroxy-lup-20(29)-en-28-oiacid) is a pentacyclictriterpene isolated from various plants including, for example,Quisqualis fructus, Coussarea paniculata, Caesalpinia paraguariensis,Vitex negundo, Ilex macropoda (Togeeswari and Sriram, Current MedicialChemistry, 2005, 12, 657-666).

Lupeol is a pharmacologically active triterpenoid having the formula(3β,13ξ)-Lup-20(29)-en-3-ol. The chemical structures of Betulin,Betulinic Acid and Lupeol are as follows:

The term “derivative”, when used in association with pentacyclictriterpenes, i.e. “pentacyclic triterpene derivative”, or any examplesof pentacyclic triterpenes such as “betulin derivatives”, or “betulinicacid derivatives” refers to a compound that is derived from thepentacyclic triterpene by a chemical or physical process. For example,pentacyclic triterpene derivatives refer to pentacyclic triterpenecompounds in which the substituents on the A-, B-, C- and D-rings aremodified in a way well known to a person skilled in the art.

Suitable betulin derivatives, betulinic acid derivatives, and related(steroid-like) compounds would be known to a person of skill in the art,and include such compounds as can be found, for example, in thefollowing documents, the contents of which are incorporated herein byreference:

WO 02/26761

Yogeeswari and Sriram, 2005, Current Medicinal Chemistry, 12, 657-666

U.S. Pat. No. 6,458,834

U.S. Pat. No. 6,403,816

U.S. Pat. Pubn Appln. 20060252733 A1

U.S. Pat. No. 6,228,850

U.S. Pat. No. 5,962,527

U.S. Pat. No. 5,869,535

U.S. Pat. No. 6,214,814

U.S. Pat. No. 6,048,847

US Pubn. Appln. 2002068098

US Pubn. Appln. 2002099164

US Publn. Appln. 2002091091

CA 2515384

DE 19854402

EP 22-1999092

JP 19-19970603

JP 17-20010619

JP 12-19970331

JP 7-19991026

JP 13-19981006

JP 7-19970311

WO 95/04526

WO 0209698

WO 99/47113

WO 99/16449

WO 00/03749

WO 00/03748

WO 02/09720 and

WO 96/29068.

By “substituent” it is meant an atom or group of atoms substituted inplace of a hydrogen atom on the parent chain of a hydrocarbon. The termssubstituent, side-chain, group, branch, or pendant group are used almostinterchangeably to describe branches from a parent structure.Combinations of substituents are permissible only if such combinationsresult in stable compounds.

The term “analogue” refers to a compound having similar chemicalproperties to the referenced compound.

The term “PEGylation” is meant to refer to the act of covalentlycoupling a PEG molecule to a compound, for example, a pentacyclictriterpene, which is then referred to as a PEGylated pentacyclictriterpene. The compounds described herein are “bis” pentacyclictriterpenes. As used herein, the term “bis” refers to two pentacyclictriterpenes conjugated to a single PEG molecule. To couple a PEG to twopentacyclic triterpenes it is necessary to activate the PEG by preparinga derivative of the PEG having a functional group at at least twotermini. In general, a functional group is chosen based on the type ofavailable active group on the molecule to which it is to be coupled. APEG molecule may be coupled to a betulin molecule through either of itsterminal hydroxyl groups. In some embodiments the linkage between thePEG molecule and a pentacyclic triterpene may be a carboxylic esterlinkage. However, a person of skill in the art would understand thatother linkages are possible. For example, the alcohol of a pentacyclictriterpene could by conjugated to PEG via a carbonate linkage (OCOOPEG),a carbamate linkage (OCONHPEG), or a phosphate ester linkage. Techniquesused to activate PEGs and form PEG derivatives would be known to aperson of skill in the art.

In embodiments wherein the pentacyclic triterpene is Betulinic acid, anacid on the PEG molecule must be activated before coupling to theBetulinic Acid. Acid halides or esters of N-hydroxysuciccinimide andpentafluorophenol, or other leaving groups could be employed in thistransformation. In other embodiments, the acid group of Betulinic acidmay be coupled directly to the PEG via an ester bond. In a furtherembodiment, the linkages are the combination of various covalent bondsincluding, but not limited to, carboxylic ester, carbonate andcarbamate. In other embodiments, the PEG molecule may be covalentlybound using mixed functional linkages.

As used herein, the term “polyethylene glycol (PEG)” refers to thecompound H(OCH₂CH₂)_(n)OH which typically exists in polymer chains. PEGis also known as polyethylene oxide (PEO) or polyoxythylene (POE),depending on its molecular weight. PEGs are prepared by polymerizationof ethylene oxide and are commercially available over a wide range ofmolecular weights from 300 g/mol to 10,000,000 g/mol. Most PEGs includemolecules with a distribution of molecular weights (i.e. they arepolydisperse). The size distribution can be characterized statisticallyby its weight average molecular weight (Mw) and its number averagemolecular weight (Mn), the ratio of which is called the polydispersityindex (Mw/Mn). MW and Mn can be measured by mass spectrometry. As usedherein, the numbers that are included in the names of PEGs indicatetheir average molecular weights. For example, a PEG with n=9 would havean average molecular weight of approximately 400 daltons (Da), and maybe labeled PEG 400. PEG molecules can range in molecular weight from 200Da to 40000 Da or more.

In some embodiments the PEG may have a molecular weight of about 1500 Dato about 10000 Da. In some embodiments, the PEG may have a molecularweight in the range of 1500 Da to 8000Da. In some embodiments the PEGmay have a molecular weight of 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000Da, 8000 Da, 9000 Da or 10000 Da. In an embodiment, the PEG has amolecular weight of about 3000 Da. In a further embodiment, the PEG hasa molecular weight of about 4000 Da.

The PEG molecules disclosed herein are capable of binding at least twopentacyclic triterpene molecules. The PEG molecule may be capable ofbinding more than two pentacyclic triterpene molecules.

In some embodiments the PEG molecules are bifunctional PEG derivatives.As used herein the term “bifunctional PEG derivatives” refers to a PEGmolecule that has been functionalized with a reactive moiety at twoterminals and is capable of conjugating at least two pentacyclictriterpene molecules.

In some embodiments, the PEG molecules may be homobifunctionalderivatives. As used herein, the term “homobifunctional poly(ethyleneglycol) (PEG)” refers to PEG derivatives that are synthetic polyetherscontaining two of the same type of functional groups. The functionalgroup may be, for example, a carboxylate, a carbamate, a carbonate, anamine, an azide, an alkyne, an alkene, a sulfonyl chloride, a phosphonylchloride, a maleimide, NHS esters, a pentafluorophenyl ester, athioester, acrylates, methacrylates, carboxylates and thiols. Examplesof homobifunctional PEG derivatives include, but are not limited to, α,ωbiscarboxymethyl PEG,α,ω-Bis{2-[(3-carboxy-1-oxopropyl)amino]ethyl}polyethylene glycol,4,7,10,13,16,19,22,25,32,35,38,41,44,47,50,53-Hexadecaoxa-28,29-dithiahexapentacontanedioicacid, O,O′-Bis(2-aminoethyl)polyethylene glycol,O,O′-Bis(2-azidoethyl)polyethylene glycol, or Poly(ethylene glycol)bis(carboxymethyl) ether.

In a preferred embodiment, the PEGylated bis pentacyclic triterpenes areesters. In an embodiment, the reactive moieties on the PEG molecule arecarboxylic acid, which form an ester linkage with the OH groups presentat C3 or C28 of a first pentacyclic triterpene and C3 or C28 of a secondpentacyclic triterpene. In an embodiment the ester linkage is formedbetween the OH group present at C3 of a first pentacyclic triterpenemolecule and the OH group present at C28 of a second pentacyclictriterpene molecule. In another embodiment the ester linkage is formedbetween the OH group present at C3 of a first pentacyclic triterpenemolecule and the OH group present at C3 of a second pentacyclictriterpene molecule. In yet another embodiment the ester linkage isformed between the OH group present at C28 of a first pentacyclictriterpene molecule and the OH group present at C3 of a secondpentacyclic triterpene molecule. In a further embodiment the esterlinkage is formed between the OH group present at C28 of a firstpentacyclic triterpene molecule and the OH group present at C28 of asecond pentacyclic triterpene molecule. In an embodiment, the PEGylatedbis pentacyclic triterpenes esters are PEGylated bis betulin esters.

In a preferred example, the homobifunctional PEG molecule is apolyethylene glycol bis(caboxymethyl) ether, also known as polyethyleneglycol 250 diacid, having the formula HOOCCH₂(OCH₂CH₂)_(n)OCH₂COOH andCAS ID 39927-08-7.

In some embodiments, the PEG molecules are heterobifunctionalderivatives which are PEGs bearing dissimilar terminal groups. As usedherein, the term “heterobifunctional poly(ethylene glycol) (PEG)derivatives” are synthetic polyethers containing two functional groupsof different types.

The PEG molecules may have different geometries. For example, the PEGmolecule may be linear or may be branched, multiarm, forked, orY-shaped. Branched PEGs may have three to ten PEG chains emanating froma central core group. Star PEGs may have 10 to 100 PEG chains emanatingfrom a central core group. Comb PEGs may have multiple PEG chainsnormally grafted onto a polymer backbone.

In some embodiments, the compounds and compositions disclosed herein areuseful in treating or preventing a fungal disease in a plant.

As used herein, “treating” means either slowing, stopping or reversingthe progression of the disease. In a preferred embodiment, “treating”means reversing the disease to the point of eliminating the disease. Insome embodiments, “treating” refers to protecting uninfected tissues andnew growth and does not refer to promoting sick tissues (yellow or brownleaves, rotted roots) to become healthy again.

As used herein “preventing” means either slowing, stopping or reversinga fungal disease in plants that has not yet occurred. The plant may beat risk of developing fungal disease due to growth conditions or nearbyinfection.

Methods are described herein for treating a plant showing symptoms of adisease or for preventing a disease in a plant that is at risk ofdeveloping a pathogenic infection. Such methods comprise administering acompound or a composition as described herein to the plant, a plant partor parts (such as, but not limited to a leaf, a stem, a trunk, or aroot) or soil supporting the plant in an effective amount. The soilsupporting the plant is the soil that physically supports the plant andfrom which the plant draws its nutrients. In some embodiments thepathogenic infection may be an infection caused by a fungal orfungal-like organism.

An “effective amount” of a PEGylated compound, with respect to thesubject method of treatment, refers to an amount of the PEGylatedcompound, which when applied inhibits or brings about, e.g. prevents orproduces changes in the rate or number symptoms of the disease accordingto acceptable standards for the disease to be treated or the effectdesired.

The compounds and compositions disclosed herein may be delivered hourly,daily, weekly, monthly, yearly (e.g. in a time release format) or as aone-time delivery. In certain embodiments, the PEGylated compounds areadministered at a dose per plant per day of at least about 0.8 g/L, in 5doses over a period of 7 days. In certain embodiments the the PEGylatedcompounds may be administered at a dose per plant per day of about 0.15g/300 mL. In other embodiments the PEGylated compounds may beadministered at a dose of 0.8 g/L to about 10 g/L per plant. In furtherembodiments the PEGylated compounds may be administered at a range of0.15-1.1 g/300 mL per plant. When used on a preventive basis, compoundsand compositions may be used at lower rates and/or at longer intervalsbetween applications. Conversely, when used to treat a disease thecompounds and compositions may be used at higher rates and/or at shorterintervals. A person of skill in the art would understand how to modifythe effective amount accordingly.

It is understood that one skilled in the art that the dose of thecomposition may vary depending on the plant type, the growth conditionsand the type of administration. It is routine in the art to adjust theeffective amount to suit the type of plant, the type of pathogen and theenvironmental conditions.

The term “plant pathogen” is meant to include any pathogen that caninfect a plant and cause disease. The plant pathogen may be, forexample, a bacteria, a fungus or a fungus-like pathogen. A fungus refersto a distinct group of eukaryotic spore-forming organisms withabsorptive nutrition and lacking chlorophyll. It includes mushrooms,molds and yeasts.

Fungal or fungal-like pathogens often cause disease which exhibit“blight” as a symptom. Blight refers to a rapid and complete chlorosis,browning, then death of plant tissues such as leaves, branches, twigs,or floral organs. Accordingly, many diseases that primarily exhibit thissymptom are called blights. Several notable examples of diseases thatexhibit blight as a symptom are: late blight of potato, caused by thewater mold Phytophthora infestans, Southern corn leaf blight, caused bythe fungus Cochliobolus heterostrophus, Chestnut blight, caused by thefungus Cryphonectria parasitica, early blight of potato and tomato,caused by species of the ubiquitous fungal genus Alternaria and leafblight of grasses. On leaf tissue, symptoms of blight include theinitial appearance of lesions which rapidly engulf surrounding tissue.

Late blight attacks a wide range of potato varieties and is one of themost destructive diseases of potato in Canada and worldwide. It iscaused by Phytophthora infestans, an oomycete (fungal-like organism).Late blight occurs in all areas of the country, but it is more severeunder high and frequent rainfall, high humidity and cool to moderatetemperatures. Early symptoms of late blight first appear on leaves assmall, circular or irregularly shaped, dark, water-soaked lesions thatcan occur within 3 to 5 days of the initial infection. On petioles andstems, symptoms appear as dark, water-soaked lesions. Lesions expand asthe pathogen colonizes the internal plant tissues. On mature lesions,the pathogen produces white, spore-bearing structures called sporangiaon the underside of the leaf spots or on the surface of diseased stems.As the disease progresses, the entire vegetative/leafy part of the plantdecays and becomes necrotic. Tubers can become infected at any stage oftheir development. Infected tubers exhibit sunken lesions on the surfaceand a reddish-brown rot is visible under the skin. Infected tubers aresusceptible to secondary infection by other pathogens, such as soft rotbacteria, that result in a soft, watery decay of the tubers.

Botrytis blight, also known as gray mold, is a fungal disease caused byseveral species in the genus Botrytis. Botrytis blight causes buds andflowers to develop abnormally and turn brown. Affected parts may becovered with a gray mold following damp, cool weather. It affects thebuds, flowers, leaves, and bulbs of many plants including: Africanviolet, begonia, chrysanthemum, cyclamen, dahlia, geranium, lily, peony,rose, and tulip. Botrytis cineria is a necrotrophic fungus that affectsmany plant species including but not limited to soft fruits; grapes,strawberries, blueberries, tomatoes, and bulb crops; carrots, parsnips,beets, beans, turnips, onions and garlic. The extent and severitydepends on weather conditions and cultural practices.

Blight is also a disease of turfgrass. Microdochium nivale (pink snowmould in turfgrass, also causes Michrodium patch), Botrytis cinerea(bean blight, gray mold other plants). The causative organism of thesediseases. Microdochium nivale, was formerly known as Fusarium nivale andMichrodium patch is sometimes referred to as Fusarium patch. Thecompounds and compositions disclosed herein are useful in the treatmentof blight.

The fungal disease may be a disease caused by a fungal pathogen selectedfrom the group consisting of Phytophthora infestans, Phytophthorainfestans and Botrytis cinerea.

Synthesis of PEGylated bis-pentacyclic triterpenes

The compounds described herein may be prepared using methods known tothose of skill in the art. The following synthetic route provides onepossible route for formation of a PEGylated bis pentacyclic triterpenehaving an ester linkage, but it is not intended as limiting, as otherroutes are possible.

Generally, the method involves reacting a minimum of two stoichiometricequivalents of a pentacyclic triterpene with a bifunctional PEG in asuitable organic solvent such as toluene or tetrahydrofuran, in thepresence of a condensing reagent, such as N,N′-Dissopropylcarbodiimide(DIC) and, if required, an acyl transfer catalyst, such as4-Dimethylaminopyridine (DMAP) to form a PEGylated bis pentacyclictriterpene ester.

In a preferred embodiment, the compound has the Formula I:

wherein P is a bifunctional PEG molecule. The bifunctional PEG moleculemay be attached via an ester linkage at positions C3 or C28 of a firstbetulin molecule and C3 or C28 of a second betulin molecule.

The compounds and compositions disclosed herein may be formulated as asprayable formulation, a wettable powder, a flowable, a dry flowable, awater-dispersible granule, or an emulsifiable concentrate. However,these formulations are not limiting and a person of skill in the artwould understand how to formulate the compounds and compositionsdisclosed herein for application to plants.

The PEGylated bis pentacyclic triterpene esters disclosed herein may beused in combination with known fungicidal products in the sameformulation. Appropriate fungicides would be known to a person of skillin the art. For example, the PEGylated bis pentacyclic triterpene estersdisclosed herein may be prepared in prepackaged mixtures containing twoor more active ingredients. Mixtures may provide protection againstfungicide resistance and may provide a broader spectrum of activityagainst fungal plant diseases. Also, improved disease control(synergism) may occur with mixtures of fungicides. Prepackaged mixturesoffer convenience and assurance against incompatibility.

In an embodiment, the PEGylated bis pentacyclic triterpenes disclosedherein may be used in combination with mono PEGylated pentacyclictriterpenes. Formulations comprising PEG Betulin mono ester with PEG Bisbetulin ester produce a mixture with increased amounts of the active(PEGylated bis betulin) form when compared to formulations comprisingPEG Bis betulin ester alone.

EXAMPLE 1

Synthesis of PEG Betulin Mono Ester Analogues

Three derivatives of PEG Betulin Mono Ester were prepared. The first twoderivatives were synthesized by esterifying the free carboxylate of PEGBetulin Mono Ester with either ethanol or octanol (Scheme 1.) The thirdderivative was prepared from carboxylmethyl MPEG 3,000 Da (Scheme 2.) toproduce an MPEG Betulin Ester. In this derivative the free carboxylicacid group of PEG Betulin mono ester is replaced with a methyl ether.

PEG Betulin Mono Ester 3,500 Da (10 g) was combined with toluene (150mL) and heated to reflux. The distillate (50 mL) was removed via a deanstark trap and the mixture was then cooled to 30° C. To the mixture wasthen added DMAP (203 mg, 0.5 equiv.) followed by octanol (4.6 mL, 10equiv) and DIC (0.57 mL, 1.0 equiv.). The mixture was then stirred for 2hours and isolated by pouring the mixture into stirring MTBE (500 mL).After cooling in an ice bath the solid was collected, bulk washed withisopropyl alcohol for 1 hour, then collected and dried. Yield: 7.9 g.

EXAMPLE 2

Solubility of PEG Betulin Esters in Aqueous Solution

The aqueous solubility of the PEG betulin derivate esters produced inEXAMPLE 1 in aqueous solution was then determined. The results of thistesting is shown in Table 1.

TABLE 1 Solubility of PEG Betulin Esters in aqueous solution. Aqueoussolubility Compound (g/L) PEG Betulin Monoester >400 PEG Mono BetulinEthyl Ester >400 PEG Mono Betulin Octyl Ester >300 MPEG Betulin Ester>400

The results in Table 1 show that the presence of the free carboxylicacid group in PEG Betulin Mono Ester does not impart a significantaqueous solubility when compared to other derivatives. Materials bearingeither an ethyl or octyl ester at the other end of the polymer, as wellas a methyl ether, exhibited similar high aqueous solubility.

EXAMPLE 3

Synthesis of PEG bis-Betulin Ester 4,000 Da and PEG bis-Betulin Ester7,000 Da

PEG bis-betulin ester 4000 Da was prepared according to Scheme 3:

Scheme 3. Synthesis of PEG bis-Betulin Ester 4,000 Da

PEG bis betulin ester 4,000 Da was prepared using PEG Bis-acid 3,000 Da.PEG Bis-Acid 3,000 Da (20 g) was combined with toluene (600 mL) andheated to reflux. The distillate (100 mL) was removed via a dean starktrap and the mixture was then cooled to 50° C. To the mixture was thenadded DMAP (407 mg, 0.5 equiv.) followed by Betulin (3.24 g, 1.1 equiv)and DIC (1.03 mL, 1.0 equiv.). The mixture was then stirred for 2 hoursand isolated by pouring the mixture into stirring MTBE (1.0 L). Aftercooling in an ice bath the solid was collected and dried. Yield: 21.1 g.

EXAMPLE 4

Solubility of PEG Betulin Esters in Aqueous Solution at AmbientTemperature and in Octanol at 37° C.

The aqueous solubility of PEG Betulin derivative esters produced inEXAMPLEs 1 and 3 was determined at ambient temperature, while thesolubility in octanol was determined at 37° C. Table 2 shows thesolubility of PEG betulin esters in aqueous solution at ambienttemperature and in octanol at 37° C.

TABLE 2 Solubility of PEG Betulin Esters Aqueous Octanol Molecularsolubility solubility Weight Of at Ambient at 37° C. Compound Compound(g/L) (g/L) PEG Betulin Monoester 3,500 Da >400 ~20 PEG Bis BetulinEster 4,000 Da ~1.6 >400 PEG Mono Betulin Ethyl Ester 3,500 Da >400 ~250PEG Mono Betulin Octyl Ester 3,500 Da >300 >300 MPEG Betulin Ester 3,500Da >400 ~8 g/L PEG Betulin Monoester 10,500 Da >400 <10 PEG Bis BetulinEster 7,000 Da ~2.4 NA PEG Bis Betulin Ester 11,000 Da >400 <10

The solubility of PEG Betulin Mono Ester 3,500 Da in water was found tobe significantly higher than that of PEG bis-Betulin Ester. An uppersolubility limit was not determined during testing, however miscibilitywas observed at up to 400 g/L or about 50 g of dissolved Betulin/L ofaqueous solution. A usable solution of PEG Betulin Mono Ester wasdetermined to be approximately 200 g/L or about 25 g of dissolvedBetulin/L of aqueous solution.

In addition to the significant increase in solubility of themono-PEGylated Betulin, the synthesis of this compound produced amixture that was highly enriched in mono-PEGylated Betulin (77%) withsurprisingly low levels of PEG bis-Betulin Ester. A furtherinvestigation identified reaction temperature as a critical processparameter for increasing the selectivity of this reaction, producingmaterials with up to 80% PEG Betulin Mono Ester containing as low as 4%PEG bis-Betulin Ester. These results indicate the chemistry used toprepare these PEGylated Betulin derivatives is remarkably selectivetowards the formation of PEG Betulin Mono Ester and produces productsthat have a high solubility in aqueous solution.

In general, materials that exhibit high aqueous solubility showed poorsolubility in warm octanol. PEG Bis Betulin Ester, which is slightlysoluble in water, was found to be very soluble in warm octanol.Interestingly, both PEG Mono Betulin Ethyl and Octyl esters showed goodsolubility in both water and warm octanol, with the Octyl ester havingmeasurably higher octanol solubility than the Ethyl ester.

EXAMPLE 5

Solubility of PEG Betulin Esters Mixed in Various Ratios

Various amounts of PEG Betulin monoester and PEG Bis Betulin ester werecombined in dichloromethane and stirred until dissolved. The productswere then precipitated with MTBE, collected and dried.

The aqueous solubility of each mixture was then determined at ambienttemperature and the relative amounts of each component was thencalculated. Results are tabulated in Table 3.

TABLE 3 Solubility of PEG Betulin Esters mixed in various ratiosSolubility Mono Bis Ratio of of Mixture Dissolved Dissolved Mono:Bis(g/L) (g/L) (g/L) 100:0  >400 >400 0  0:100 ~1.6 0 ~1.6 1:1 45 22.5 22.53:1 90 67.5 22.5 9:1 175 157.5 17.5

The results in table 3 indicate the mixing PEG Bis Betulin Ester withPEG Betulin mono Ester can improve the overall solubility of PEG BisBetulin ester. These findings indicate that a formulation with a higherdosage of PEG Bis Betulin Ester can be prepared. In some embodiments theformulation of PEG Betulin mono ester with the PEG Bis betulin estercould produce a mixture with up to 10× the amount of the active formwhen compared to formulating PEG Bis betulin ester alone. A higherdose/L could be applied to a plant when such mixtures are prepared.

EXAMPLE 6

Efficacy of PEG-BETULIN for Control of Late Blight of Potato and CropTolerance, Under Greenhouse Conditions.

In this example, weekly applications of PEG bis-betulin andPEG-mono-betulin at various concentrations were assessed for control ofPhytophthora infestans on the susceptible potato cultivar ‘Colomba’compared to a commercial standard fungicide Copper Spray 50W (copperoxychloride), an inoculated (water) check and a non-inoculated (healthy)check. The trial was conducted in winter in a research greenhouse.

It was postulated that a more soluble form of PEGylated Betulin wouldallow for a higher dose of antifungal agent per unit of applied solutionand thusly produce greater antifungal activity. In order to investigatethis hypothesis, a mono-PEGylated form of Betulin was prepared by usingonly one stoichiometric equivalent of Betulin during the couplingreaction with PEG bis-Acid (Scheme 2.).

Materials:

PEG (polyethylene glycol), PEG-bis-betulin ester (4.0 kDa) as preparedin Scheme 3, PEG-mono-BETULIN (3.5 kDa) as prepared in Scheme 1, COPPERSPRAY 50W (copper oxychloride, 50%).

Methodology:

The trial was conducted in a designated research poly greenhouse atKwantlen Polytechnic University, Langley, BC, from Dec. to March. OnDecember 23, 100×2-gallon pots were washed with dish soap and sanitizedby dipping in CHEMPROCIDE (7.5% DDAC) at 10 mL/L for 5 minutes. Thepotting medium consisted of 2/3 Sunshine Mix #4 and ⅓ coarse perlite.The greenhouse had supplemental light and heat. An overhead mistingsystem was set up to provide irrigation and the research bench wascovered with an irrigation capillary mat throughout the trial period, tomaintain high humidity for infection and disease development.

The potatoes were seeded on December 30, Osmocote™ Plus 15-9-12 6-monthslow release granular fertilizer (with micronutrients) was added to themoist potting mixture at 65 g per pot and mixed thoroughly by hand.Sprouting potato tubers cv. ‘Colomba’ (obtained from H. Niven, ESCropconsult Ltd., Richmond, BC) were cut to include at least two eyesper seed piece, placed 5-6 inches deep into the potting mix and lightlycovered with the potting mix. The misting system was turned back on andtimer was set to run for 2 min every 6 h beginning at 6 pm. Soiltemperature at planting was 15° C. and soil moisture was approximately20%. Shoot emergence after 2 weeks was 99%. The seed pieces were nottreated with any fungicide or insecticide.

ENTONEM (Steinernema feltiae nematodes, Koppert Biologicals) was appliedas a drench at 5000 nematodes per mL in 50 ml per pot on January 14, tocontrol fungus gnats. No fungus gnats were present in the followingweeks.

Two days before the first test product application, shoot tips were cutback by 10-15 cm to encourage bushing out and to remove any lanky orovergrown shoots. Pruners were wiped with alcohol between cuts but thecuts were allowed to air dry and were not sprayed with disinfectant. Theplants were tied and staked to prevent breakage of shoots.

Applications: The trial was laid out in a randomized complete block(RCB) design on a single greenhouse bench with 5 replicates pertreatment and 2 plants per plot (10 plants per treatment). The testproducts were applied 5 times at weekly intervals, February 5, 12, 19,26 and March 6 and, on each application date, the check plots weresprayed with water alone. The solution volume applied was equivalent to1500 L/ha (300 mL/2 m²). For each application, the plants in eachtreatment were removed from the trial bench, placed in a 2 m² area on aplastic ground sheet, sprayed with the product or water, then returnedto the bench, after the product had dried. Test product applications andrates are summarized in Table 4.

TABLE 4 Treatments, rates and volume applied. Solution Solution Volume/Amount of Rate of No. of Volume Treatment Product/ Product Applicationsper (per 2 m² Mix Volume Treatment Applied and Interval hectarespray-area) (300 mL) 1. Un-inoculated — — 1500 L 300 mL — (Healthy)Check (water only) 2. Inoculated Check — — 1500 L 300 mL — (water only)3. PEG only 2.8 g/L 5 × 7 days 1500 L 300 mL 0.56 g 4. PEG-bis-BETULIN0.8 g/L 5 × 7 days 1500 L 300 mL 0.15 g 4.0 KDa at ½ X rate 5.PEG-bis-BETULIN 1.6 g/L 5 × 7 days 1500 L 300 mL 0.32 g 4.0 KDa at 1 Xrate 6. COPPER SPRAY  4.0 g/L* 5 × 7 days 1500 L 300 mL  1.2 g 50W(standard label rate)* 7. PEG-mono- 0.7 g/L 5 × 7 days 1500 L 300 mL0.14 g BETULIN 3.5 KDa at ¼ X rate 8. PEG-mono- 1.4 g/L 5 × 7 days 1500L 300 mL 0.27 g BETULIN 3.5 KDa at ½ X rate 9. PEG-mono- 2.8 g/L 5 × 7days 1500 L 300 mL 0.56 g BETULIN 3.5 KDa at 1 X rate 10. PEG-mono- 5.6g/L 5 × 7 days 1500 L 300 1.11 BETULIN 3.5 KDa at 2 X rate

After each application, the trial area was covered with a clear plastictent and the irrigation was turned off for 24 h to allow the products todry on the leaves. After the second last application on February 26, theoverhead mist was turned off and the plants were sub-irrigated only, bywetting the capillary mat with two soaker hoses, for 6 min. twice a day,at 10 a.m. and 10 p.m.

Inoculation: Inoculum of Phytophthora infestans (genotype US8 originallyisolated from a potato crop in the BC Fraser Valley and obtained fromDr. L. Kawchuk, AAFC, Lethbridge, AB) was applied on February 13, 24 hafter the second fungicide application in a total volume of 1.5 L ofinoculum solution per 90 plants. The inoculum was prepared from 30,28-day-old, V8 agar plates (200 mL clarified V8 juice, 2.0 g CaCO₃, 0.05g B-cholesterol/L). Each plate was flooded with 10 mL sterile dH₂O andmycelium and sporangia were gently scraped with a scalpel into a 1 Lflask. This solution was poured through a #18 mesh strainer to removemycelial clumps. The total volume of 300 mL contained 6.3×10⁴ sporangiaper mL (mean of 3 haemocytometer slides×2 counts each). To this wasadded 250 mL of a 5-day-old liquid culture in the same V8 medium thathad been incubated on a shaker at 100 rpm in the dark at roomtemperature and contained 3×10⁶ zoospores per mL by haemocytometercount. Distilled water was added to bring the final volume up to 1.5. L.The final inoculum solution contained a calculated concentration of2.1×10⁴ sporangia/mL and 5×10⁶ zoospores/mL.

The plants were inoculated using a pump-action broadcast sprayer overthe entire plant area. The inoculated plants were covered with blackplastic for 24 h to create high humidity and low light for sporangialgermination and infection of the plant tissue. The non-inoculated checkplants were placed 5 m away on a separate bench for protection from thespores in the inoculation mixture, and covered with black plastic, also.After 24 h, the black plastic was removed and replaced with a clearplastic tent and the mist irrigation was turned back on. The ends of theplastic tent were left open to allow air movement and cooling on sunnydays. After the first product application, the non-inoculated, watercheck plants were returned to the trial bench in the RBC design.

A preliminary inoculation test on potato and tomato plants showed thatlate blight lesions developed from either sporangia (solid plate) orzoospore (liquid culture) inoculum after 10 days under the trialconditions. Lesions were produced on both potato cv. ‘Colomba’ and ontomato plants in a KPU student trial on a nearby bench.

Disease Assessment: The number of P. infestans lesions per plant (leavesand petioles) and the percentage of diseased leaves out of total leavesper plant was counted weekly, from one week after the first application(February 12; 24 h prior to inoculation) to one week after the lastapplication (March 12). Disease severity was assessed using theHorsfall-Barratt grade scale of 0-11, a standard assessment scale forlate blight of potato. The H-B grades were transformed to percentagesfollowing the accepted formula of Redman, King and Brown (ELANCO). Thearea under the disease progress curve (AUDPC) was calculated for eachassessment parameter over the course of the trial where N=the number ofdisease assessments; t=time; y (0) is the initial infection or diseaselevel at t=0 (i.e., when disease was first observed), y_(i) is thedisease rating on each date and the AUDPC (A_(k)) at t=t_(k), is thetotal accumulated disease rating at the end of the trial: ²

$A_{k} = {\sum\limits_{i = 1}^{N_{i} - 1}\; {\frac{\left( {y_{i} + y_{i + 1}} \right)}{2}\left( {t_{i + 1} - t_{i}} \right)}}$

Data was analyzed statistically (ANOVA) using CoStat, Version 6.400,2008, CoHort Software, Monterey Calif., USA, ©1998-2008, and treatmentmeans were compared in LSD, Duncan's Multiple Range Test (Duncan's MRT)and Tukey's HSD at P=0.05. LSD is the weakest statistical test, Duncan'sMRT is mid-range and Tukey's is the strongest test.

Environmental conditions in the greenhouse were recorded at eachapplication time and temperature and relative humidity were recordedhourly, throughout the trial period, using a HOBO data logger placed onthe bench in the crop canopy. Environmental conditions in the greenhousewere cool and humid, with a mean temperature of 12.6-17.6° C. and a meanrelative humidity of 80.9-96.7, favourable for development of lateblight of potato caused by P. infestans (see Table 5).

TABLE 5 Mean hourly recordings: HOBO Data Logger. Temperature (° C.)Relative Humidity Max- Max- Month imum Minimum Mean imum Minimum MeanDecember 30-31 19.7 16.5 17.6 99.5 75.7 90.8 January 1-31 27.6 7.3 16.098.5 48.2 80.9 February 1-28 26.8 0.1 12.6 100 52.0 93.4 March 1-12 32.14.2 14.8 100 34.2 96.7

FIG. 1 shows the disease incidence, i.e., the mean number of late blightlesions per plant per date and area under the disease progress curve(AUDPC) over the course of the trial and is summarized in Table 3.

As can be seen in Table 6, mean number of late blight lesions per plant(Table 6) there was no statistical difference between the inoculatedcheck and the treated plants until March 5, when the treatments withPEG-bis-betulin at 0.5 and 1X rates and Copper 50W had fewer diseaselesions than the inoculated check in LSD at P=0.05. On March 12, oneweek after the last application, plants treated with PEG bis-betulin atthe 1X rate had a mean of 8.5 lesions per plant, statistically differentfrom the check in LSD at P=0.05, while plants treated with Copper 50Whad a mean of 5.5 lesions per plant, statistically different from theinoculated check in Duncan's MRT at P=0.05, but not statisticallydifferent from PEG bis-betulin at the 1X rate. Overall, in AUDPC, theplants treated with PEG-betulin at the 1X rate had 60.4% fewer lateblight lesions than the inoculated check. The 0.5X rate ofPEG-bis-betulin, PEG alone and the mono-betulin formulations were notstatistically different from the inoculated check.

TABLE 6 Mean number of late blight lesions per plant.^(1,2) TreatmentProduct Rate Feb 12³ Feb 19³ Feb 26³ Mar 5³ Mar 12³ AUDPC³ 1. InoculatedWater 0.2 ± 0.2 ± 2.4 ± 14.2 ± 19.0 ± 184.8 ± Check Only 0.6 ab 0.6 bc1.6 abc 15.9 a 12.5 a 155.6 a (a) (ab) (ab) (a) (a) (a) 2. Non- Water0.0 ± 0.0 ± 0.0 ± 0.7 ± 5.0 ± 22.4 ± Inoculated Only 0.0 b (a) 0.0 c (b)0.0 c (b) 1.4 c (b) 5.3 c (c) 28.0 c (b) Check [a] 3. PEG only 2.8 g/L0.5 ± 0.7 ± 3.1 ± 13.3 ± 18.3 ± 185.5 ± 1.1 ab 1.3 abc 3.2 ab 12.7 a10.8 a 130.0 a (a) (ab) (a) (a) (a) (a) 4. PEG-bis- 0.8 g/L 0.0 ± 1.6 ±2.2 ± 4.7 ± 11.4 ± 99.4 ± betulin 0.5X 0.0 b (a) 3.1 abc 2.1 abc 5.2 bc8.9 abc 70.0 abc (ab) (ab) (ab) (abc) (ab) 5. PEG-bis- 1.6 g/L 0.2 ± 0.6± 1.0 ± 4.5 ± 8.5 ± 73.2 ± betulin 1X 0.4 ab 0.9 bc 1.1 bc 5.3 bc 8.7 bc66.9 bc (a) (ab) (ab) (ab) (abc) (ab) 6. COPPER 4.0 g/L 2.3 ± 2.3 ± 3.8± 4.2 ± 5.5 ± 99.4 ± 50W 5.6 a (a) 4.3 ab 4.5 a (a) 4.9 bc 4.3 c 126.2(ab) (ab) (bc) abc (ab) 7. PEG- 0.7 g/L 0.5 ± 0.7 ± 2.5 ± 9.9 ± 15.9 ±149.1 ± mono- 0.8 ab 0.9 abc 1.9 abc 8.4 ab 12.9 ab 102.9 ab betulin (a)(ab) (ab) (ab) (ab) (a) 0.25X 8. PEG- 1.4 g/L 1.4 ± 1.9 ± 3.4 ± 11.4 ±12.7 ± 166.2 ± mono- 2.4 ab 2.6 abc 4.2 ab 13.8 ab 17.0 abc 198.0 abbetulin 0.5X (a) (ab) (a) (a) (abc) (a) 9. PEG- 2.8 g/L 0.2 ± 1.1 ± 3.1± 7.7 ± 13.7 ± 132.0 ± mono- 0.6 ab 2.3 abc 4.1 ab 9.1 abc 9.1 abc 115.1ab betulin 1X (a) (ab) (a) (ab) (abc) (ab) 10. PEG- 5.6 g/L 2.1 ± 2.9 ±3.8 ± 10.4 ± 14.1 ± 176.4 ± mono- 4.3 ab 4.9 a (a) 4.5 a (a) 10.1 ab 9.1abc 158.3 ab betulin 2X (a) (ab) (abc) (a) ¹Mean and standard deviationof two plants per plot, 5 replicates per treatment, RCB design.²Treatments applied Feb. 5, Feb. 12, Feb. 19, Feb. 26 and March 5.³Numbers in the same column followed by the same letter are notsignificantly different in LSD and (Duncan's MRT) in brackets at P =0.05. No differences were found in Tukey's HSD at P = 0.05.

FIG. 2 (and Table 7) show the mean percentage of diseased leaves andAUDPC. On March 5, both of the PEG-bis-betulin treatments, had asignificantly lower percentage of diseased leaves than the inoculatedcheck, different in Duncan's MRT at P=0.05, and were similar to thecommercial fungicide, COPPER 50W (Table 7). Over all of the trial, theplants treated with PEG bis-betulin at the 1X rate had 57% fewerdiseased leaves than the inoculated check, significantly different inDuncan's at P=0.05 and not statistically different from COPPER 50W(Table 7).

TABLE 7 Mean percentage of diseased leaves per plant.^(1,2) TreatmentProduct Rate Feb 12³ Feb 19³ Feb 26³ Mar 5³ Mar 12³ AUDPC³ 1. InoculatedWater 1.1 ± 1.0 ± 8.8 ± 21.6 ± 32.4 ± 337.7 ± Check Only 3.5 ab 3.0 ab6.7 a 26.0 a 24.6 a 284.2 a (a) (a) (a) 2. Non- Water 0.0 ± 0.0 ± 0.0 ±1.0 ± 9.1 ± 38.7 ± Inoculated Only 0.0 b 0.0 b 0.0 b 2.0 e 7.4 bc 38.3 dCheck (c) (a) (b) 3. PEG only 2.8 g/L 3.2 ± 2.8 ± 6.9 ± 19.4 ± 30.9 ±324.9 ± 8.0 ab 5.2 ab 8.3 a 14.5 17.6 ab 160.6 a ab (a) (a) (ab) 4.PEG-bis- 0.8 g/L 0.0 ± 7.8 ± 6.9 ± 8.2 ± 20.8 ± 232.9 ± betulin 0.0 b15.7 a 6.1 a 5.6 17.1 abc 153.4 0.5X bcde (a) abc (ab) (abc) 5. PEG-bis-1.6 g/L 1.2 ± 2.2 ± 3.5 ± 5.5 ± 17.9 ± 145.3 ± betulin 1X 2.5 ab 3.2 ab3.6 ab 5.3 26.6 abc 140.3 cde (a) bcd (ab) (abc) 6. COPPER 4.0 g/L 2.1 ±2.2 ± 4.1 ± 4.5 ± 5.9 ± 103.5 ± 50W 3.4 ab 3.2 ab 3.6 ab 3.8 de 2.7 c78.4 cd (bc) (a) (ab) 7. PEG- 0.7 g/L 2.5 ± 2.1 ± 6.2 ± 16.0 ± 28.1 ±277.8 ± mono- 4.3 ab 2.9 ab 3.3 a 10.0 28.9 ab 144.0 ab betulin abc (a)(ab) 0.25X (abc) 8. PEG- 1.4 g/L 4.8 ± 6.3 ± 8.4 ± 11.2 ± 14.1 ± 248.9 ±mono- 5.5 ab 4.8 ab 5.3 a 8.5 14.9 abc 153.6 betulin abcde (a) abc (ab)0.5X (abc) 9. PEG- 2.8 g/L 1.4 ± 3.2 ± 7.1 ± 10.8 ± 32.1 ± 265.3 ± mono-4.5 ab 7.5 ab 7.0 a 9.5 38.0 abc 242.4 betulin 1X abcde (a) abc (ab)(abc) 10. PEG- 5.6 g/L 5.4 ± 5.8 ± 7.7 ± 15.2 ± 19.7 ± 289.1 ± mono-10.3 a 7.7 ab 6.2 a 6.7 8.9 abc 187.9 ab betulin 2X abcd (a) (a) (abc)¹Mean and standard deviation of two plants per plot, 5 replicates pertreatment, RCB design. ²Treatments applied Feb. 5, Feb. 12, Feb. 19,Feb. 26 and March 5. ³Numbers in the same column followed by the sameletter are not significantly different in Duncan's MRT and (Tukey's HSD)in brackets at P = 0.05.

Disease severity, i.e., the mean percent leaf diseased per plant andAUDPC is shown in Table 8 and FIG. 3. On March 12, the plants treatedwith PEG-bis-betulin at the 1X rate also had the lowest percentage ofleaf area diseased (except for the non-inoculated (healthy) check)(Table 8).

TABLE 8 Mean percentage of leaf area diseased per plant (rated on theH-B scale transformed to percentages).^(1,2) Treatment Product Rate Feb12³ Feb 19³ Feb 26³ Mar 5³ Mar 12³ AUDPC³ 1. Inoculated Water 1.2 ± 1.2± 3.4 ± 5.8 ± 22.1 ± 155.9 ± Check Only 0.3 b 0.3 b 2.4 b 4.9 a 23.8 a128.9 (a) (a) abc (a) 2. Non- Water 1.2 ± 1.2 ± 1.2 ± 1.4 ± 2.8 ± 40.1 ±Inoculated Only 0.0 b 0.0 b 0.0 b 0.4 c 0.9 d (b) 6.9 c (a) Check (a) 3.PEG 2.8 g/L 1.4 ± 1.8 ± 3.6 ± 7.4 ± 16.1 ± 151.7 ± only 0.5 b 1.1 b 3.2b 6.5 a 12.8 abc 98.2 abc (a) (ab) (a) 4. PEG-bis- 0.8 g/L 1.1 ± 1.5 ±2.4 ± 2.2 ± 9.6 ± 81.1 ± betulin 0.0 b 0.5 b 0.9 b 0.3 a 10.1 bcd 36.5abc 0.5X (a) (ab) (a) 5. PEG-bis- 1.6 g/L 1.4 ± 1.6 ± 1.8 ± 2.1 ± 4.3 ±59.4 ± betulin 1X 0.5 b 0.6 b 0.6 b 1.0 a 2.9 cd 17.2 bc (a) (b) (a) 6.COPPER 4.0 g/L 3.4 ± 6.1 ± 8.1 ± 8.3 ± 5.9 ± 190.2 ± 50W 5.5 a 11.3 a11.7 a 11.6 a 5.3 cd 264.9 a (a) (ab) (a) 7. PEG- 0.7 g/L 1.5 ± 1.6 ±3.6 ± 7.8 ± 20.0 ± 167.2 ± mono- 0.56 b 0.6 b 2.4 b 5.0 a 13.7 ab 76.0ab betulin (a) (ab) (a) 0.25X 8. PEG- 1.4 g/L 2.0 ± 2.3 ± 3.5 ± 7.9 ±15.4 ± 157.8 ± mono- 1.1 ab 1.0 b 2.3 b 7.5 a 16.0 abc 122.0 betulin (a)(ab) abc (a) 0.5X 9. PEG- 2.8 g/L 1.5 ± 1.4 ± 3.7 ± 6.5 ± 9.6 ± 120.9 ±mono- 1.10 b 0.5 b 5.3 b 10.9 a 10.1 bcd 147.8 betulin 1X (a) (ab) abc(a) 10. PEG- 5.6 g/L 2.0 ± 2.3 ± 2.7 ± 8.1 ± 8.4 ± 129.1 ± mono- 1.5 ab1.4 b 1.1 b 10.6 a 6.0 bcd 83.1 abc betulin 2X (a) (ab) (a) ¹Mean andstandard deviation of two plants per plot, 5 replicates per treatment,RCB design. ²Treatments applied Feb. 5, Feb. 12, Feb. 19, Feb. 26 andMarch 5. ³Numbers in the same column followed by the same letter are notsignificantly different in Duncan's MRT and (Tukey's HSD) in brackets atP = 0.05.

The mean percentage of diseased leaf are per plant was equivalent inplants treated with PEG-bis-betulin 1X and plants treated with COPPER50W and the only treatment statistically different from the inoculatedcheck in Tukey's HSD at P=0.5. Overall, the AUDPC for leaf area diseasedwas 62% less than the inoculated check and statistically lower thancopper. On March 12, the highest rates (1X and 2X) of PEG-mono-betulinwere statistically better than the control also, i.e., had smallerdisease lesions than the water control, and were statistically similarto the commercial fungicide treatment, but not in overall AUDPC.

FIG. 4 shows the mean percentage of leaf area diseased each week of thetrial (rated on the H-B scale transformed to percentages). A few diseaselesions appeared naturally on the plants prior to inoculation onFebruary 13; possibly from a student experiment inoculated the previousweek. The non-inoculated plants remained disease-free until March 5,which confirmed that the disease was actively spreading among thetreated plants under the plastic tent. Disease lesions increased greatlyin size in the last week (percent leaf area diseased). No phytotoxicitywas observed in any treatment.

CONCLUSIONS

In a replicated, randomized greenhouse experiment, on potato cv.‘Colomba’ inoculated with a US8 strain of Phytophthora infestans, weeklyfoliar sprays of PEG bis-betulin at the 1X rate (1.6 g/L) suppressedlate blight of potato as well as, or better than, the registeredfungicide Copper 50W (copper oxychloride) at the label rate (4 g/L).This PEG bis-betulin treatment reduced the number of disease lesions by60%, the number of infected leaves by 57% and leaf area affected by 62%compared to the inoculated control, in AUDPC. The 0.5X dilution of thePEG-bis betulin product gave some disease suppression, but wasstatistically ineffective.

The PEG-mono-betulin formulation gave only slight disease suppressionand was not statistically effective at any concentration, from 0.25X to2X (0.7-5.6 g/L). However, at the 1X rate, PEG mono-betulin reduced thenumber of disease lesions by 28.6%, the number of infected leaves by21.4% and leaf area affected by 22.5% compared to the inoculatedcontrol, in AUDPC. PEG alone had no effect on disease pressure.

The greenhouse plants were irrigated by overhead irrigation twice dailyfor the first 3 weeks of the trial and only sub-irrigated for the finaltwo weeks. Irrigation did not appear to affect disease development orproduct performance, except for the standard fungicide, COPPER 50W,which performed better without overhead irrigation. One of thelimitations of copper fungicide is that it is easily dissolved in waterand washes off of the leaves under rain or irrigation. No phytotoxicitywas observed in any treatment.

Without being bound by theory, these results indicate the combination ofhigh lipid solubility and low water solubility of PEG Bis Betulin Estercompared to PEG mono betulin may be responsible for its increasedantifungal activity, as compared to PEG mono betulin.

EXAMPLE 6

PEG-bis-betulin was tested for control of Fusarium patch (pink mould) ofturfgrass caused by Microdochium nivale in fall/winter.

An aqueous solution of PEG bis-Betulin Ester, as prepared in Example 3was also tested for use as an antifungal agent against Microdochiumpatch (pink snow mould) on golf green turf grass. On a commercial golfgreen, PEG bis-betulin formulated at a 1X rate suppressed the disease aswell as the registered fungicides BANNER MAXX (propiconazole) or COMPASS50WG (trifloxystrobin) at their lowest label rates. Three applicationsof PEG bis-betulin in the fall reduced the number of disease spots by57% and the percent turf area diseased by 70% compared to the control,in February of the following year. The 0.5X dilution of the PEG-betulinproduct was ineffective. A 2X application of the product, i.e., twicethe solution volume, increased the incidence and severity of the diseasesomewhat compared to the 1X rate, although there were no visiblesymptoms of phytotoxicity.

PEG bis-Betulin was found to be significantly different from the watercontrol for all parameters tested and not statistically different whencompared to the standard commercial fungicides, BANNER MAXX and COMPASS50 WG. There was, however, no significant difference in disease levelsamong the rates of PEG-betulin applied.

EXAMPLE 7

PEG-bis-betulin was tested for control of Botrytis of bean in agreenhouse crop in Winter.

A solution of PEG-bis-Betulin Ester 4,000 Da (1.7 g/L) in water, asprepared in EXAMPLE 3 was applied preventatively as a foliar spray 24hours before inoculation of bean leaves with a known fungal pathogen,Botrytis cinerea. This application reduced the number of Botrytislesions on bean leaves for up to 14 days and performed as well as thestandard botrytis fungicide, SWITCH 62.5 WDG (cyprodinil+fludioxonil).The number of lesions was 50% less than on the inoculated check plantsand the maximum lesion size was also significantly reduced. The percentleaf area diseased was 90.7% less than the inoculated check after 14days, compared to 63.5% for plants treated with SWITCH fungicide. Nophytotoxicity was observed in any treatment.

When applied three days post-inoculation, as a “curative” spray to treatinfected plants, PEG bis-Betulin Ester appeared to stop lesionexpansion: the mean number and maximum diameter of botrytis lesions inthe PEG-betulin “curative” treatment were statistically less than in theinoculated check for up to 7 days after inoculation. The percent leafarea diseased was 81.4% less than the check at 14 days. No phytotoxicitywas observed in any treatment.

PEG-bis-betulin at a concentration of 1.7 g/L showed both protectantactivity when applied 24-48 hours pre-inoculation and curative activitywhen applied 72 hours post-inoculation.

In other testing, systemic activity was observed, in that upper leavesdeveloped fewer and smaller lesions when PEG-betulin was applied to thelower leaves only. This may be due to induction of natural plant defensemechanisms (SAR) or translocation of the product systemically within theplant. However, disease control with the product “in planta” wasachieved at a 1/5 dilution of the effective rate on Botrytis cinerea “invitro”, which suggests a possible stimulation of the plant's own defensemechanisms (SAR) in addition to a direct effect on the pathogen. Nophytotoxicity was observed.

Without being bound to theory, it is presumed that this water solubleform of betulin is readily absorbed by the leaves of the test plant.Once absorbed, the ester bond of the PEG bis-betulin ester prodrug maybe hydrolyzed chemically or enzymatically, resulting in the release ofbetulin. After separation from the PEG carrier, betulin may then be freeto function as an antifungal agent.

EXAMPLE 8

Laboratory Tests in-vitro: Botrytis cinerea

PEG-bis-betulin as prepared in EXAMPLE 3 inhibited fungal mycelialgrowth in vitro. When Botrytis cinerea and nutrients for fungal growthwere added to the liquid formulation of PEG-bis-betulin at the 1X rate,no difference was found in the number of Botrytis colonies plated after48 hours, but, after 7 days, a 77.9% reduction in fungal weight(Botrytis mycelial growth) was recorded as compared to the water check.PEG-bis-betulin at a 50 or 75% dilution was not different from the watercheck.

The above-described embodiments are intended to be examples only.Alterations, modifications and variations can be effected to theparticular embodiments by those of skill in the art. The scope of theclaims should not be limited by the particular embodiments set forthherein, but should be construed in a manner consistent with thespecification as a whole. The contents of all references and publishedpatent applications cited throughout this application are herebyincorporated by reference.

What is claimed is:
 1. A PEGylated bis pentacyclic triterpene having the formula: A-P-B, wherein, A is a first pentacyclic triterpene; B is a second pentacyclic triterpene; and P is a polyethylene glycol (PEG) molecule, for use in preventing or treating a fungal disease in a plant.
 2. The PEGylated bis pentacyclic triterpene of claim 1, wherein the first and second pentacyclic triterpenes are each independently betulin, betulinic acid, lupeol, or an analogue or derivative thereof.
 3. The PEGylated bis pentacyclic triterpene of claim 1, wherein the first and second pentacyclic triterpenes are each betulin.
 4. The PEGylated bis pentacyclic triterpene of claim 1, wherein the PEG molecule has a molecular weight of about 1500 Da to about 8,000 Da.
 5. The PEGylated bis pentacyclic triterpene of claim 4, wherein the PEG molecule has a molecular weight of about 3,000 Da.
 6. The PEGylated bis pentacyclic triterpene of claim 1, wherein the PEG molecule is α,ω biscarboxymethyl PEG.
 7. The PEGylated bis pentacyclic triterpene of claim 3, wherein the PEG molecule is covalently bound via a first ester linkage to C3 or C28 of the first pentacyclic triterpene and via a second ester linkage to C3 or C28 of the second pentacyclic triterpene.
 8. The PEGylated bis pentacyclic triterpene of claim 1, wherein the fungal disease is blight.
 9. The PEGylated bis pentacyclic triterpene of claim 1, wherein the fungal disease is a disease caused by a fungal pathogen selected from the group consisting of Phytophthora infestans, Phytophthora infestans and Botrytis cinerea.
 10. A fungicidal composition comprising the PEGylated bis pentacyclic triterpene as defined in claim 1; and an agriculturally acceptable diluent.
 11. A method of treating or preventing a fungal disease in a plant comprising: applying to the plant, to a soil supporting the plant, or to both the plant and the soil supporting the plant, a composition comprising: a PEGylated bis pentacyclic triterpene having the formula: A-P-B, wherein, A is a first pentacyclic triterpene; B is a second pentacyclic triterpene; and P is a polyethylene glycol (PEG) molecule; and an agriculturally acceptable diluent.
 12. The method of claim 11, wherein the composition is applied to the leaves of the plant.
 13. The method of claim 11, wherein the first and second pentacyclic triterpenes are each independently betulin, betulinic acid, lupeol, or an analogue or derivative thereof.
 14. The method of claim 11, wherein the first and second pentacyclic triterpene are each betulin.
 15. The method of claim 11, wherein the PEG molecule has a molecular weight of about 1500 Da to about 8,000 Da.
 16. The method of claim 15, wherein the PEG molecule has a molecular weight of about 3,000 Da.
 17. The method of claim 11, wherein the PEG molecule is α,ω biscarboxymethyl PEG.
 18. The method of claim 14, wherein the PEG molecule is covalently bound via a first ester linkage to C3 or C28 of the first pentacyclic triterpene and via a second ester linkage to C3 or C28 of the second pentacyclic triterpene.
 19. The method of claim 11, wherein the fungal disease is blight.
 20. The method of claim 19, wherein the fungal disease is a disease caused by a fungal pathogen selected from the group consisting of Phytophthora infestans, Phytophthora infestans and Botrytis cinerea. 